http://www.translational-medicine.com/content/pdf/1479-5876-11-262.pdf

Peng et al. Journal of Translational Medicine 2013, 11:262
http://www.translational-medicine.com/content/11/1/262
RESEARCH
Open Access
Comprehensive analysis of the percentage of
surface receptors and cytotoxic granules positive
natural killer cells in patients with pancreatic
cancer, gastric cancer, and colorectal cancer
Yun-Peng Peng1,2†, Yi Zhu1,2†, Jing-Jing Zhang1,2†, Ze-Kuan Xu1,2, Zhu-Yin Qian1,2, Cun-Cai Dai1,2, Kui-Rong Jiang1,2,
Jun-Li Wu1,2, Wen-Tao Gao1,2, Qiang Li1,2, Qing Du1,2 and Yi Miao1,2*
Abstract
Background: Digestive malignancies, especially pancreatic cancer (PC), gastric cancer (GC), and colorectal cancer
(CRC), still occur at persistently high rates, and disease progression in these cancers has been associated with tumor
immunosurveillance escape. Natural killer (NK) cell dysfunction may be responsible for this phenomenon, however,
the exact relationship between tumor immunosurveillance escape in digestive malignancies and NK cell
dysfunction remains unclear.
Methods: Percentage of the surface receptors NKG2A, KIR3DL1, NKG2D, NKp30, NKp44, NKp46, and DNAM-1, as
well as the cytotoxic granules perforin and granzyme B positive NK cells were determined in patients with
pancreatic cancer (n = 31), gastric cancer (n = 31), and CRC (n = 32) prior to surgery and healthy controls (n = 31) by
multicolor flow cytometry. Independent t-tests or Mann-Whitney U-tests were used to compare the differences
between the patient and healthy control groups, as well as the differences between patients with different
pathologic features of cancer.
Results: Percentage of NKG2D, NKp30, NKp46, and perforin positive NK cells was significantly down-regulated in
patients with PC compared to healthy controls, as well as GC and CRC; reduced levels of these molecules was
associated with indicators of disease progression in each malignancy (such as histological grade, depth of invasion,
lymph node metastasis). On the contrary, percentage of KIR3DL1 positive NK cells was significantly increased in
patients with PC, as well as GC and CRC, but was not associated with any indicators of disease progression.
Conclusions: Altered percentage of surface receptors and cytotoxic granules positive NK cells may play a vital role
in tumor immunosurveillance escape by inducing NK cell dysfunction in patients with PC, GC, and CRC.
Keywords: Cytotoxic granules, Digestive malignancies, NK cells, Surface receptors
* Correspondence: [email protected]
†
Equal contributors
1
Department of General Surgery, The first Affiliated Hospital of Nanjing
Medical University, 300 Guangzhou Road, Nanjing 210029, People’s Republic
of China
2
Jiangsu Province Academy of Clinical Medicine, Institute of Tumor Biology,
300 Guangzhou Road, Nanjing 210029, People’s Republic of China
© 2013 Peng et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Peng et al. Journal of Translational Medicine 2013, 11:262
http://www.translational-medicine.com/content/11/1/262
Background
Pancreatic cancer, gastric cancer and colorectal cancer are
the most common digestive malignancies and have relatively high incidences. Pancreatic cancer is characterized
by a low rate of early diagnosis and many tumors are
unresectable [1], with a 5-year survival rate of only 6% [2]
leading to a persistently high rate of mortality [3]. Colorectal cancer and gastric cancer are the third and fourth
most common cancers worldwide, respectively, and are
among the leading causes of cancer-related deaths [1]. In
humans, the progression of certain malignancies is associated with the immune function of certain lymphocytes,
such as natural killer (NK) cells. NK cells are CD16- and/
or CD56-positive, and represent the first line of immune
defense against transformed malignant cells [4].
When infection or malignancy occur, circulating NK
cells become activated by cytokines and infiltrate into the
affected tissues containing pathogen-infected or transformed malignant cells [5]. The direct cytotoxic effects of
NK cells are determined by their expression of surface receptors and cytotoxic granules. NK cell dysfunction is observed in patients with certain types of cancer; therefore,
surface receptors and cytotoxic granules are an important
area of cancer research.
The natural cytotoxicity receptors (NCRs) NKp30,
NKp44, and NKp46 are expressed on NK cells, as well as
T cells and NK-like cells [6-9], and mediate NK cell activation during the process of natural cytotoxicity. Killer cell
lectin-like receptor subfamily K, member 1 (NKG2D),
a C-type lectin-like protein, is an activating receptor
expressed on NK cells and also gamma-delta T cells,
natural killer T (NKT) cells and other types of immune
cells [10]. NKG2D is required for the ability of NK cells to
lyse harmful cells [11,12]. NK cells also express other activating receptors including DNAX accessory molecule-1
(DNAM-1) which binds to two well-characterized ligands
(nectin-2 and the poliovirus receptor) and exerts similar
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effects to NKG2D [13]. Killer cell lectin-like receptor subfamily C, member 1 (CD94/NKG2A-B), killer cell lectinlike receptor subfamily C, member 2 (CD94/NKG2C-E)
and the killer immunoglobulin-like receptors (KIRs) are
described as inhibitory receptors, which are important for
the education of NK cells and NK-induced cytotoxicity
through interacting with the major histocompatibility
complex (MHC) class I allotype [14]. The cytotoxic granules perforin and granzyme B are intracellular molecules
present in a number of lymphocytes, including NK cells.
Perforin is required for the ability of granzyme B to promote apoptosis in target cells [15,16]. NK cells express
high levels of perforin and granzyme B, and the expression
levels of these molecules are closely associated with the
cytotoxicity of NK cells [17].
Methods
Patients and healthy controls
Patients diagnosed with PC (n = 31), GC (n = 31), or CRC
(n = 32) who were treated at Jiangsu Province Hospital
were enrolled in this study. All patients had only received
positive preoperative preparation and had not undergone
radiotherapy, chemotherapy or any other therapeutic
strategies prior to surgery. The main clinicopathological
features of the patient cohorts are shown in Table 1. All
peripheral blood samples were collected from the patients
before surgery, and peripheral blood samples from 31
healthy control individuals were provided by Jiangsu
Province Blood Center. This study was approved by
the Ethics Committee of the First Affiliated Hospital of
Nanjing Medical University. Each of the patients and
healthy control individuals gave informed consent.
Reagents
The anti-human CD3-FITC/CD16 + 56-PE mixed antibody
was obtained from Beckman Coulter (Brea, CA, USA).
The anti-human CD3-FITC, CD16-PE/Cy7, CD56-PE/Cy7,
Table 1 Clinicopathological features of the patients and healthy controls included in this study
Clinicopathological characteristics
Groups
Gender
Age
AJCC Stage*
*
Healthy controls
Pancreatic cancer
Gastric cancer
n = 31
n = 31
n = 31
Colorectal cancer
n = 32
Male
21(67.7%)
23(74.2%)
20(64.5%)
17(53.1%)
Female
10(32.3%)
8(26.8%)
11(35.5%)
15(46.9%)
Median age
53
64
61
60
Range
35-57
34-76
35-82
40-82
0
1(3.2%)
0
I
0
7(22.6%)
1(3.1%)
II
20(64.5%)
4(12.9%)
16(50.0%)
III
0
17(54.8%)
14(43.8%)
IV
11(35.5%)
2(6.5%)
1(3.1%)
0
2010 American Joint Committee on Cancer (AJCC).
Peng et al. Journal of Translational Medicine 2013, 11:262
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NKG2D-PE/Cy7, NKp44-APC, NKp46-PE/Cy7, NKp30APC, KIR3DL1-PE, DNAM-1-Alexa Fluor 647, and
perforin-PerCP/Cy5.5 antibodies, and the RBC Lysis Buffer, Fixation Buffer and Wash Buffer were purchased from
Biolegend (San Diego, CA, USA), as well as FITC, PE, PE/
Cy7, APC, PerCP, Alexa Fluor-647, and PerCP/Cy 5.5
mouse IgG1 antibodies. The anti-human NKG2A-PerCP
and granzyme B-APC antibodies were obtained from
R&D Systems (Minneapolis, MI, UAS). All antibodies
were mouse monoclonal antibodies.
Preparation of peripheral blood samples and flow
cytometric analysis
Each peripheral blood sample (2 ml) was aliquoted into
four tubes (100 μl per tube), which were labeled tube-1,
tube-2, tube-3 and tube-4, respectively.
Peripheral blood samples of tube-1, tube-2 and tube-3
were stained to detect surface receptors as follows. Firstly,
to identify NK cells, anti-human CD3-FITC/CD16 + 56PE mixed antibodies were added to tube-1 and tube-2.
Anti-human CD3-FITC, CD16-PE/Cy7 and CD56-PE/Cy7
antibodies were added to tube-3. Secondly, anti-human
NKG2D-PE/Cy7 and NKp44-APC antibodies were added
to tube-1. Anti-human NKG2A-PerCP, NKp46-PE/Cy7
and NKp30-APC antibodies were added to tube-2. Antihuman KIR3DL1-PE and DNAM-1-Alexa Fluor-647 antibodies were added to tube-3. The three tubes were
incubated in the dark at room temperature for 15-20 min.
Then 2 ml RBC Lysis Buffer was added per tube. After
incubating in the dark at room temperature for 15 min,
the cells were washed twice with PBS.
Peripheral blood sample of Tube-4 was stained to detect cytotoxic granules as follows. Firstly, anti-human
CD3-FITC/CD16 + 56-PE mixed antibodies were added
to tube-4 to identify NK cells. After incubating in the
dark at room temperature for 15-20 min, 2 ml RBC
Lysis Buffer was added per tube, and the mixtures were
incubated in the dark at room temperature for 15 min.
Then the cells were washed twice with PBS and fixation
Buffer (500 μl per tube) was added. The mixtures were
incubated in the dark at room temperature for 20 min,
and then the cells were washed twice with Wash Buffer.
Lastly, anti-human perforin-PerCP/Cy5.5 and granzyme
B-APC antibodies were added to tube-3. After incubating in the dark at room temperature for 15 min, the
cells were washed twice with PBS.
Flow cytometric analysis
According to cell physical characteristics, forward scatter
(FSC) and side scatter (SSC), a cell subset located in left
lower quadrant (PBMCs) was selected from total cell
subset and defined as gating “A”. And then, according to
cells staining, another cell subset which detected as
CD3-/CD(16 + 56) + (NK cells) was selected from gating
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“A” and defined as gating “Q”. Further detections for
surface receptors and cytotoxic granules were based on
cells from gating “Q”. The whole detection for per tube
would stop until getting 10000 cells from gating “Q”.
Isotype control was applied in our study to exclude nonspecific fluorescence using matched isotype monoclonal
antibodies (FITC, PE, PE/Cy7, and APC mouse IgG1
antibodies for tube 1; FITC, PE, PE/Cy7, PerCP, and
APC mouse IgG1 antibodies for tube 2; FITC, PE, PE/
Cy7, and Alexa Fluor-647 antibodies for tube 3; FITC,
PE, PerCP/Cy 5.5, and APC mouse IgG1 antibodies for
tube 4). Data were detected by multicolor flow cytometry (Gallios, Beckman Coulter, Brea, CA, USA) and
gallios software (Beckman Coulter, Brea, CA, USA), and
analyzed by Kaluza software (Beckman Coulter, Brea,
CA, USA).
Statistical analysis
Independent t-tests were used to compare the differences
between two groups when the two groups both accorded
with normal distribution, otherwise Mann–Whitney Utests were used. Independent t-tests and Mann–Whitney
U-tests were performed using Statistical Product and
Service Solutions 19.0 (SPSS 19.0) (SPSS Inc., Chicago, IL,
USA). Data were expressed as means ± standard deviations (Mean ± SD). The level of statistical significance accepted was P < 0.05.
Results
Percentage of surface receptor and cytotoxic granule
positive circulating NK cells
We determined the percentage of seven surface receptors
positive circulating NK cells in both healthy controls and
patients with PC, GC, and CRC by multicolor flow cytometry. The percentage of tested molecules positive circulating NK cells of the cancer patients and healthy controls
are presented in Figure 1 and Table 2.
Compared to the healthy controls, significantly decreased
levels of activating receptors NKG2D, NKp30, NKp46,
and DNAM-1 positive NK cells were observed in PC
patients (P < 0.001, P < 0.001, P < 0.001, and P < 0.01, respectively); however, an significantly increased level of
inhibitory receptor KIR3DL1 positive NK cells was observed in patients with PC (P < 0.001). In GC patients, the
activating receptors NKG2D, NKp30, and NKp46 positive
NK cells were also significantly down-regulated compared
to the healthy controls (P < 0.001, P < 0.001, and P < 0.001,
respectively); however, the inhibitory receptor KIR3DL1
positive NK cells was also significantly up-regulated compared to the healthy controls (P < 0.001). Furthermore, the
levels of activating receptors NKG2D, NKp30, and NKp46
positive NK cells in CRC patients was significantly lower
compared to healthy controls (P < 0.01, P < 0.001, and
P < 0.001, respectively); however, the level of inhibitory
Peng et al. Journal of Translational Medicine 2013, 11:262
http://www.translational-medicine.com/content/11/1/262
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Figure 1 Distribution of the percentage of surface receptor and cytotoxic granule positive circulating NK cells in healthy controls
(Controls) and patients with pancreatic cancer (PC), gastric cancer (GC), and colorectal cancer (CRC). The bar for each group was means
and standard deviations (Mean and SD).
receptor KIR3DL1 positive NK cells was also significantly
higher compared to the healthy controls (P < 0.001).
We also determined the percentage of cytotoxic perforin and granzyme B positive circulating NK cells in
both healthy controls and patients with PC, GC, and
CRC (Figure 1 and Table 2). Respectively compared to
the healthy controls, percentage of perforin positive NK
cells was significantly lower in patients with PC, GC,
and CRC (P < 0.01, P < 0.001, and P < 0.001, respectively).
Percentage of granzyme B positive NK cells was at high
levels in both the NK cells of the patients with cancer
and the healthy controls.
Altered percentage of NKG2D, NKp30, NKp46, KIR3DL1,
and perforin positive NK cells correlate with disease
progression
The correlations between the percentage of NKG2D,
NKp30, NKp46, KIR3DL1, and perforin positive NK cells
and the pathologic features of PC, GC, and CRC are respectively shown in Tables 3, 4 and 5.
In pancreatic cancer, NKG2D, NKp30, NKp46, KIR3DL1,
and perforin had no association with the presence of
distant metastasis. In non-metastatic pancreatic cancer,
the percentage of NKG2D and NKp30 positive NK cells
were significantly decreased in patients with lymph
Peng et al. Journal of Translational Medicine 2013, 11:262
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Table 2 Respective comparison of the percentage of surface receptors and cytotoxic granules positive NK cells in
healthy controls and three kinds of cancer patients
Healthy controls
Pancreatic cancer
Gastric cancer
Colorectal cancer
%
%
P
%
P
%
P
NKG2A
1.5 ± 1.0
1.9 ± 1.0
ns U
1.3 ± 1.2
ns U
1.4 ± 1.0
ns U
NKG2D
92.1 ± 2.7
83.4 ± 8.4
< 0.001 T
86.9 ± 2.9
< 0.001 T
84.9 ± 10.0
< 0.01 U
U
42.6 ± 19.4
< 0.001 U
0.9 ± 1.3
ns U
55.1 ± 24.0
< 0.001 U
89.5 ± 5.1
ns U
19.5 ± 13.1
< 0.001 U
97.4 ± 2.5
ns U
76.3 ± 19.1
< 0.001 U
NKp30
71.8 ± 15.3
46.5 ± 20.2
NKp44
1.8 ± 0.9
2.4 ± 1.7
< 0.001
U
ns U
1.7 ± 1.4
U
NKp46
84.8 ± 5.1
56.4 ± 22.2
< 0.001
DNAM-1
91.4 ± 4.6
87.2 ± 5.5
< 0.01 U
KIR3DL1
9.6 ± 2.7
19.8 ± 11.4
< 0.001
Granzyme B
97.9 ± 1.6
96.9 ± 2.4
ns U
Perforin
U
95.2 ± 3.0
79.9 ± 16.0
< 0.01
36.6 ± 21.3
U
U
< 0.001
ns U
50.1 ± 29.5
< 0.001
87.7 ± 7.8
ns U
17.9 ± 12.3
< 0.001
97.8 ± 2.0
ns U
81.2 ± 15.5
< 0.001
U
U
U
represented Mann–Whitney U-tests and T represented independent t-tests. Data were expressed as means ± standard deviations (Mean ± SD).
node metastasis than patients without lymph node
metastasis (both P < 0.05). The levels of NKG2D and
perforin positive NK cells were significantly lower in
patients with blood vessel invasion, compared to patients with non-metastatic pancreatic cancer who did
not have blood vessel invasion (P < 0.05 and P < 0.01).
NKp46 positive NK cells percentage also correlated
closely with the histological grade in non-metastatic
pancreatic cancer (P < 0.01).
In gastric cancer, the percentage of NKG2D, NKp30,
and perforin positive NK cells were significantly lower in
patients with lymph node metastasis than patients without
lymph node metastasis (P < 0.01, P < 0.05 and P < 0.05,
respectively). NKG2D positive NK cells were significantly down-regulated in patients with blood vessel
invasion compared to patients without blood vessel invasion (P < 0.05). NKG2D, NKp30, and perforin positive
NK cells were significantly higher levels in patients with
gastric cancer who had well or moderately differentiated
tumors, compared to those with poorly differentiated
tumors (P < 0.01, P < 0.05, and P < 0.05, respectively).
Moreover, the percentage of NKp30 positive NK cells
correlated significantly with the depth of invasion in
gastric cancer (P < 0.05).
In colorectal cancer, NKG2D, NKp46, and perforin
positive NK cells were significantly lower levels in
patients with lymph node metastasis compared to
patients without lymph node metastasis (P < 0.01, P < 0.05,
and P < 0.01). The percentage of NKp30, NKp46, and
perforin positive NK cells correlated markedly with
depth of invasion in CRC (all P < 0.05). The percentage of NKG2D and perforin positive NK cells correlated closely with histological grade in CRC (P < 0.01
and P < 0.05). None of the molecules tested were
associated with blood vessel invasion or nerve invasion
in CRC.
Discussion
In this study, we quantified the percentage of several
activating and inhibitory surface receptors positive
circulating NK cells, as well as the cytotoxic granules
perforin and granzyme B, in patients with PC, GC,
and CRC. The balance between activating and inhibitory receptors has been shown to be a key factor
which determines NK cell activity [18]. It has been
demonstrated that NK-mediated anti-tumor immunity
is frequently defective in patients with certain malignancies [19,20]. This study indicates that patients
with PC, GC, and CRC have dysfunctional NK cells;
therefore, NK cell dysfunction may be an important
component of tumor escape from immunosurveillance in
these cancers.
NKp30, NKp44, and NKp46 are the most well characterized NCRs. Our results show for the first time that
the numbers of NKp30 and NKp46-positive NK cells
were significantly reduced in almost all patients with PC,
GC, and CRC, consistent with studies in other malignancies such as cervical cancer, breast cancer, and melanoma [21-23]. It has been reported that NCR-positive
NK cells have the ability to kill harmful cells, such as
transformed malignant cells and infected cells, and can
also secret inflammatory cytokines such as interferon-γ
(IFN-γ) and tumor necrosis factor-α (TNF-α) [24]. Accordingly, the lower number of cells expressing NKp30
and NKp46 may be partly responsible for the poor function of NK cells in patients with PC, GC, and CRC. The
NCR-mediated interaction between NK cells and their
target cells is ligand-dependent. Cellular heparin or
heparin sulfate proteoglycans, which are expressed at
high levels on cancer cells, are ligands for all NCRs [25],
while natural killer cell cytotoxicity receptor 3 ligand 1
(B7-H6) and BCL2-associated athanogene 6 (BAT3) are
specific ligands for NKp30 [26,27]. The specific ligands
Pancreatic cancer
NO. of patients
NKG2D
%
NKp30
P
%
NKp46
P
%
Perforin
P
KIR3DL1
%
P
%
P
80.5 ± 16.8
ns U
20.6 ± 12.1
ns U
Distant metastasis
Absent
20
82.6 ± 9.5
Present
11
85.5 ± 6.4
ns
T
44.2 ± 19.3
ns
T
50.7 ± 22.1
53.8 ± 23.4
ns
T
60.9 ± 20.2
78.7 ± 15.0
18.4 ± 10.3
Non-metastatic Pancreatic cancer
NO. of patients
NKG2D
NKp30
%
P
%
ns T
46.2 ± 27.1
NKp46
P
Perforin
KIR3DL1
%
P
%
P
%
P
71.4 ± 17.4
< 0.01 U
85.3 ± 8.5
ns U
23.2 ± 13.6
ns U
Peng et al. Journal of Translational Medicine 2013, 11:262
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Table 3 Association between the percentage of surface receptors and cytotoxic granules positive NK cells and the clinicopathological features of
pancreatic cancer
Histological grade
Well/Moderately
9
85.4 ± 8.9
Poorly
11
79.6 ± 9.1
Tis/T1/T2
11
84.3 ± 7.6
T3
9
79.7 ± 10.9
Absent
6
89.9 ± 5.6
Present
14
78.9 ± 8.6
Absent
13
86.7 ± 6.7
Present
7
73.9 ± 7.6
Absent
6
86.9 ± 5.7
Present
14
80.2 ± 9.9
ns
T
40.6 ± 12.4
39.5 ± 17.1
76.5 ± 21.0
18.6 ± 10.8
Depth of invasion*
ns
T
41.2 ± 18.2
ns
T
46.0 ± 22.9
54.4 ± 22.6
ns
T
53.2 ± 25.6
75.2 ± 19.1
ns U
86.9 ± 11.6
18.7 ± 10.4
ns T
23.0 ± 14.1
Lymph node metastasis
< 0.05 T
58.1 ± 19.0
< 0.05 T
37.8 ± 17.7
60.9 ± 22.6
ns
T
50.8 ± 23.8
80.9 ± 21.7
ns U
80.3 ± 15.3
27.0 ± 15.5
ns T
17.9 ± 9.7
Blood vessel invasion
< 0.01 T
40.3 ± 22.7
ns
U
48.4 ± 13.7
54.7 ± 24.9
ns
U
52.2 ± 22.1
86.2 ± 12.9
< 0.05 U
69.9 ± 19.1
21.9 ± 14.4
ns U
18.3 ± 6.1
Nerve invasion
ns
T
36.2 ± 22.6
45.9 ± 19.0
ns
U
60.5 ± 27.6
51.0 ± 21.8
ns
U
78.1 ± 16.1
81.5 ± 17.6
ns U
15.9 ± 5.6
ns T
22.7 ± 13.6
*
According to 2010 American Joint Committee on Cancer (AJCC), T4 pancreatic cancer invades celiac artery or superior mesenteric artery, and is unresectable, so T4 is not enrolled in depth of invasion for nonmetastatic pancreatic cancer. Tis, T1, or T2 pancreatic cancer invades inside of the pancreas; T3 pancreatic primary cancer invades outside of the pancreas. U represented Mann–Whitney U-tests and T represented
independent t-tests. Data were expressed as means ± standard deviations (Mean ± SD).
Page 6 of 10
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Table 4 Association between the percentage of surface receptors and cytotoxic granules positive NK cells and the
clinicopathological features of gastric cancer
Gastric cancer
NO. of
patients
NKG2D
NKp30
NKp46
%
P
%
P
%
< 0.01 T
45.1 ± 24.4
< 0.05 U
56.3 ± 28.7
Perforin
P
KIR3DL1
%
P
%
P
88.5 ± 7.6
< 0.05 U
15.3 ± 5.9
ns U
Histological grade
Well/Moderately
12
89.0 ± 3.0
Poorly
19
86.2 ± 2.5
Tis/T1/T2/T3
10
87.8 ± 3.2
T4
21
87.0 ± 2.9
Absent
11
89.3 ± 2.8
Present
20
86.1 ± 2.5
Absent
17
88.3 ± 3.0
Present
14
85.9 ± 2.5
31.2 ± 17.7
ns
U
46.2 ± 30.1
76.5 ± 17.4
20.5 ± 14.3
Depth of invasion*
ns T
48.0 ± 24.3
< 0.05 T
31.1 ± 17.8
54.2 ± 29.1
ns
U
48.1 ± 29.1
85.5 ± 10.0
ns U
79.1 ± 17.3
21.4 ± 12.7
ns U
17.2 ± 11.6
Lymph node metastasis
< 0.01 T
48.2 ± 22.7
< 0.05 T
30.1 ± 17.9
57.6 ± 29.3
ns
U
46.0 ± 29.5
88.5 ± 8.1
< 0.05 U
77.1 ± 17.1
15.1 ± 5.7
ns U
20.4 ± 14.0
Blood vessel invasion
< 0.05 T
38.0 ± 23.8
ns
T
34.8 ± 18.4
49.0 ± 29.6
ns
U
51.4 ± 30.5
84.5 ± 12.0
ns U
77.1 ± 18.5
18.3 ± 10.8
ns U
18.8 ± 13.6
*
According to 2010 American Joint Committee on Cancer (AJCC), Tis, T1, T2, or T3 gastric cancer invades inside of gastric serosa; T4 gastric cancer invades outside
of gastric serosa or adjacent organs. U represented Mann–Whitney U-tests and T represented independent t-tests. Data were expressed as means ± standard
deviations (Mean ± SD).
for NKp46 are associated with different cells, for example,
Thr225 for some malignancy cells, Thr125 and Asn216 for
human β cells [28]. It has been shown that NKp30
is blocked by exosomal and soluble BCL-2-associated
athanogene-6 (BAG6) which are released by cancer cells;
however, during infection with certain viruses, soluble influenza haemagglutinin (HA) and pp65 take similar effect
to block NKp30 or NKp46. We suggest that such blocking
effects may contribute to the downregulation of NKp30
and NKp46 [24] on NK cells in patients with PC, GC and
Table 5 Association between the percentage of surface receptors and cytotoxic granules positive NK cells and the
clinicopathological features of colorectal cancer
Colorectal cancer
NO. of
patients
NKG2D
%
NKp30
P
%
NKp46
P
Perforin
%
P
56.0 ± 25.8
ns T
%
KIR3DL1
P
%
P
Histological grade
20
89.0 ± 3.7
< 0.01 U 39.5 ± 19.3
12
77.9 ± 13.3
47.8 ± 19.2
Tis/T1/T2/T3
7
88.9 ± 4.3
T4
25
83.7 ± 10.9
38.6 ± 19.2
Absent
18
89.3 ± 4.2
< 0.01 U 39.3 ± 20.2
Present
14
79.1 ± 12.4
46.9 ± 18.1
Absent
25
86.5 ± 7.8
Present
7
78.9 ± 15.0
Absent
27
84.0 ± 11.7
Present
5
84.7 ± 3.1
Well/Moderately
Poorly
ns
T
53.6 ± 21.8
83.9 ± 11.6 < 0.05 U 20.1 ± 15.9 ns U
63.5 ± 22.8
18.6 ± 6.6
*
Depth of invasion
ns
U
57.0 ± 12.4 < 0.05 T 71.4 ± 19.5
< 0.05 T
87.7 ± 13.0 < 0.05 U 20.7 ± 11.1 ns U
50.5 ± 23.5
73.1 ± 19.6
63.8 ± 24.8 < 0.05 U
86.2 ± 8.8
43.8 ± 18.1
63.5 ± 21.5
19.2 ± 13.8
Lymph node metastasis
ns U
< 0.01 U 21.2 ± 16.8 ns U
17.4 ± 5.5
Blood vessel invasion
ns
U
42.0 ± 20.1
ns U
44.6 ± 17.9
53.9 ± 24.1
ns U
59.1 ± 25.2
77.8 ± 18.2
ns U
70.7 ± 23.0
19.0 ± 14.5 ns U
21.4 ± 6.3
Nerve invasion
*
ns
U
40.9 ± 18.6
51.8 ± 23.1
ns
T
55.7 ± 23.3
51.4 ± 30.4
ns U
75.8 ± 19.7
78.8 ± 17.8
ns U
20.8 ± 13.7 ns U
12.7 ± 6.5
According to 2010 American Joint Committee on Cancer (AJCC), Tis, T1, T2, or T3 colorectal cancer invades inside of colorectal serosa or non-peritonealized
adjacent colorectal tissue; T4 gastric cancer invades outside of colorectal serosa or adjacent organs. U represents Mann–Whitney U-tests and T represents
independent t-tests. Data were expressed as means ± standard deviations (Mean ± SD).
Peng et al. Journal of Translational Medicine 2013, 11:262
http://www.translational-medicine.com/content/11/1/262
CRC; however, the exact mechanisms require further
research.
NKp30- and NKp46-mediated cytotoxicity of NK cells
are not only linked to the elimination of cancer cells, but
also to the eradication of bacterial and viral infection, and
regulation of immune homeostasis [29]. Further analysis
of our data revealed that the expression of NKp30 and
NKp46 correlated with pathological stage and histological
grade in patients with PC, GC and CRC, which indicates
that NK cell dysfunction may participate in malignant
progression in these tumor types.
NKG2D is an important activating receptor on NK
cells. In patients with cancer, NKG2D generally binds
specifically to killer cell lectin-like receptor subfamily
K, member 1 ligands (NKG2DLs) expressed on transformed malignant cells, such as MHC class I-related
molecules, MHC class I polypeptide-related sequence
A/B (MICA/MICB), and UL16-binding protein (ULBP)
[30]. The NKG2D-NKG2DL complex associates with
the hematopoietic cell signal transducer (DAP10) adaptor protein and induces the cytotoxic effects of NK cells
via the phosphatidylinositol-3 kinase (PI-3-K) pathway
[4]. A number of soluble factors, such as TGF-β and
L-kynurenine, which are secreted at high levels by
malignant cells, are effective inhibitors of NKG2Dassociated NK cell function [31,32]. However, our results
indicate that the NKG2D-mediated interaction between
NK cells and cancer cells is reduced during the development of PC, GC, and CRC, and similar results have
previously been reported in pancreatic cancer, gastric
cancer, and other types of cancer [20,33,34]. Additionally,
Guerra et al. demonstrated that NKG2D-deficient mice
exhibit defective tumor surveillance in models of spontaneous malignancy, which also supports our results [35].
It is noteworthy that not only down-regulation of NKG2D,
but also the release of NKG2DLs from the surface of
cancer cells may contribute to NK cell dysfunction and
the progression of some types of cancer [36].
Furthermore, our results also suggest that reduced expression of NKG2D and perforin by NK cells correlated
significantly with lymph node metastasis in PC, GC, and
CRC, and also correlated with histological grade in gastric
cancer and CRC. The view that perforin-dependent cytotoxicity is a crucial factor in NKG2D-mediated apoptosis
[37] is confirmed by this study. In response to infection or
cancer, the cytotoxic granule granzyme B associates with
perforin in NK cells to form a complex which is ultimately
released into the cytoplasm of the target cell and mediates
the cytotoxic effects of NK cells [15,16]. Therefore, reduced expression of perforin by NK cells in patients with
PC, GC, and CRC may form a crucial part of the mechanism of NK dysfunction in these cancers.
We also investigated NK cell inhibitory receptors
in this study. KIR3DL1, a well-characterized killer
Page 8 of 10
immunoglobulin-like receptor, binds the specific ligand
major histocompatibility complex, class I, Bw4 (HLABw4) [38]. Our results show that the expression of
KIR3DL1 by NK cells was significantly increased in patients with PC, GC, and CRC. However, this increase
did not correlate significantly with any pathological feature. Al Omar et al. reported similar result in patients
with kidney cancer and small-cell lung cancer, but not
in patients with non-small-cell lung cancer and colon
cancer [39]. Further research is required to determine
the role of KIR3DL1 in different types of cancer.
Conclusions
In conclusion, down-regulated percentage of the activating
receptors NKp30, NKp46, and NKG2D positive NK cells,
as well as the cytotoxic granule perforin, in patients with
PC, GC, and CRC may indicate that patients with these
digestive system cancers have dysfunctional NK cells.
Additionally, the percentage of these molecules positive
NK cells correlated with certain clinicopathological features. Thus, in certain malignancies, NK cell dysfunction
may potentially promote the escape of malignant cells
from immunosurveillance, and may also be a marker of
poor prognosis. Further research is required to determine
the exact mechanisms for why these molecules positive
NK cells are down-regulated in patients with digestive system cancers; such research may contribute to immunotherapy strategies to improve NK cell function in patients
with cancer.
Abbreviations
B7-H6: Natural killer cell cytotoxicity receptor 3 ligand 1; BAT3/BAG6: BCL2associated athanogene 6; CRC: Colorectal cancer; DAP10: Hematopoietic cell
signal transducer; DNAM-1: DNAX accessory molecule-1; FSC: Forward
scatter; GC: Gastric cancer; HA: Haemagglutinin; HLA-Bw4: Major
histocompatibility complex, class I, Bw4; IFN-γ: Interferon-γ; KIR3DL1: Killer cell
immunoglobulin-like receptor, three domains, long cytoplasmic tail, 1;
MHC: Major histocompatibility complex; MICA/MICB: MHC class I
polypeptide-related sequence A/B; NCRs: Natural cytotoxicity receptors;
NK cells: Natural killer cells; NK cells: Natural killer T cells; NKG2A: Killer cell
lectin-like receptor subfamily C, member 1-like; NKG2D: Killer cell lectin-like
receptor subfamily K, member 1; NKG2DLs: Killer cell lectin-like receptor
subfamily K, member 1 ligands; NKp30: Natural cytotoxicity triggering
receptor 3; NKp44: Natural cytotoxicity triggering receptor 2; NKp46: Natural
cytotoxicity triggering receptor 1; PC: Pancreatic cancer; PI-3K: Phosphatidylinositol-3 kinase; SD: Standard deviations; SPSS: Statistical
product and service solutions; SSC: Side scatter; TNF-α: Tumor necrosis
factor-α; ULBP: UL16-binding protein.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Y-PP, YZ and J-JZ carried out the studies, participated in the statistical
analysis and drafted the manuscript. Z-KX, Z-YQ, C-CD, K-RJ, J-LW, W-TG, QL
and QD participated in the sample collection and statistical analysis. YM
conceived of the study, and participated in its design and coordination and
helped to draft the manuscript. All authors read and approved the final
manuscript.
Peng et al. Journal of Translational Medicine 2013, 11:262
http://www.translational-medicine.com/content/11/1/262
Acknowledgements
This work was supported in part by the grants from the National Natural
Science Foundation of China (81170336, 81272239, 81101802, 81001079), the
Natural Science Foundation of Jiangsu Province (BK2011845), the Program
for Development of Innovative Research Team in the First Affiliated Hospital
of NJMU, the Priority Academic Program Development of Jiangsu Higher
Education Institutions (PAPD, JX10231801), and the research Special Fund For
public welfare industry of health (201202007).
Page 9 of 10
21.
22.
Received: 30 July 2013 Accepted: 9 October 2013
Published: 20 October 2013
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doi:10.1186/1479-5876-11-262
Cite this article as: Peng et al.: Comprehensive analysis of the
percentage of surface receptors and cytotoxic granules positive natural
killer cells in patients with pancreatic cancer, gastric cancer, and
colorectal cancer. Journal of Translational Medicine 2013 11:262.
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